Water transport in epididymal and ejaculated rhesus monkey (Macaca mulatta) sperm during freezing

In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macacamulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 °C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 μm with a radius of 0.40 μm and an osmotically inactive cell volume, V_b, of 0.772V_o, where V_o is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ∼22 μm⁻¹. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L_pg or L_pg[cpa] and the activation energy, E_Lp or E_Lp[cpa]) were found to range from: L_pg or L_pg[cpa] = 0.0020-0.0029 μm/min-atm; E_Lp or E_Lp[cpa]) = 10.6-18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, ; where the units of B_opt are °C/min, E_Lp or E_Lp[cpa] are kcal/mole, L_pg or L_pg[cpa] are μm/min-atm and SA/WV are μm⁻¹), we determined the optimal rates of freezing macaque sperm to be ∼23 °C/min (ejaculated sperm in the absence of CPAs), ∼29 °C/min (ejaculated sperm in the presence of glycerol), ∼24 °C/min (epididymal sperm in the absence of CPAs) and ∼24 °C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.     

Effect of cyanide in dark and light on the membrane potential and the ATP level of young and mature green tissues of higher plants

The effect of CN⁻ and N₂ on the electrical membrane potential (E_m) was compared with that of CN⁻ on the ATP levels in cotyledons of Gossypium hirsutum and in Lemna gibba L. In mature cotton tissue, CN⁻ depolarized E_m to the energy-independent diffusion potential (E_D) in the dark. In the light E_m recovered transiently. The same was observed in leaves of Nicotiana, Avena, Impatiens, Kalanchoë, and in Lemna. In contrast, in young cotton cotyledons and tobacco leaves and, to a large extent, in +sucrose-grown Lemna, E_m was depolarized to E_D also in the light in a similar way as in the dark.

In Lemna grown without sucrose, the energy-dependent component of E_m was only partially depolarized by CN⁻ in dark or light. Cyanide plus salicylhydroxamic acid completely reduced E_m to E_D, abolished respiration and photosynthesis, and severely diminished the ATP level. This suggests the operation of a CN⁻-insensitive respiration in uninjured Lemna. The initial CN⁻-induced decay of the ATP level in cotton and Lemna was more rapid than the decay of E_m. CN⁻-induced oscillations of the ATP level were followed by similar but slower oscillations of E_m. This supports the view of a general dependence of E_m on ATP. Discrepancies between inhibitor-induced changes of E_m and ATP levels are suggested to result from additional regulation of E_m by the cytoplasmatic pH value.

A comparison of E_D in young and mature cotton cotyledons in the dark and in the light suggests that in growing young cotyledons the different effect of CN⁻ in the light is due to a less effective photosynthesis together with high mitochondrial respiration. In Lemna and in mature cotton tissue, E_m in the light is maintained by noncyclic photophosphorylation and photosystem II, which is only partly inhibited by CN⁻, thus resulting in an incomplete depolarization and recovery of E_m. Complete inhibition of photosynthetic O₂ evolution and membrane depolarization by CN⁻ plus salicylhydroxamic acid are suggested to result from photooxidation.

     

Irradiance influences tea leaf (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.) photosynthesis and transpiration

Rates of net photosynthesis (P _N) and transpiration (E), and leaf temperature (T_L) of maintenance leaves of tea under plucking were affected by photosynthetic photon flux densities (PPFD) of 200–2 200 μmol m⁻² s⁻¹. P _N gradually increased with the increase of PPFD from 200 to 1 200 μmol m⁻² s⁻¹ and thereafter sharply declined. Maximum P _N was 13.95 μmol m⁻² s⁻¹ at 1 200 μmol m⁻² s⁻¹ PPFD. There was no significant variation of P _N among PPFD at 1 400–1 800 μmol m⁻² s⁻¹. Significant drop of P _N occurred at 2 000 μmol m⁻² s⁻¹. PPFD at 2 200 μmol m⁻² s⁻¹ reduced photosynthesis to 6.92 μmol m⁻² s⁻¹. PPFD had a strong correlation with T_L and E. Both T_L and E linearly increased from 200 to 2 200 μmol m⁻² s⁻¹ PPFD. T_L and E were highly correlated. The optimum T_L for maximum P _N was 26.0 °C after which P _N declined significantly. E had a positive correlation with P _N.     

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a K_d for DNA of 9.2 nm, with an off-rate constant of 0.013 s⁻¹and an on-rate constant of 14 μm⁻¹ s⁻¹. dNTP binding is rapid with K_d values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a K_d value for PP_i of 3.7 mm and a maximal rate constant of 11 s⁻¹. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s⁻¹; rapid phosphodiester bond formation occurs with a K_eq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s⁻¹, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.     

Optimum temperature policy for batch reactors performing biochemical reactions in the presence of enzyme deactivation

A necessary condition is found for the optimum temperature policy which leads to the minimum reaction time for a given final conversion of substrate in a well stirred, enzymatic batch reactor performing an enzyme-catalyzed reaction following Michaelis-Menten kinetics in the presence of first order enzyme decay. The reasoning, which is based on Euler's classical approach to variational calculus, is relevant for the predesign steps because it indicates in a simple fashion which temperature program should be followed in order to obtain the maximum advantage of existing enzyme using the type of reactor usually elected by technologists in the fine biochemistry field. In order to highlight the relevance and applicability of the work reported here, the case of optimality under isothermal operating conditions is considered and a practical example is worked out.List of Symbols C _E mol.m^–3 concentration of active enzyme - C _E ^* dimensionless counterpart of C_E - C _E,0 mol.m^–3 initial concentration of active enzyme - C _E,b mol.m^–3 final concentration of active enzyme - C _E,opt ^* optimal dimensionless counterpart of C_E - C _smol.m^–3 concentration of substrate - C _S ^Emphasis>/* dimensionless counterpart of C_S - C _S,0mol.m^–3 initial concentration of substrate - C _S,bmol.m^–3 final concentration of substrate - E enzyme in active form - E ₃ ^* dimensionless counterpart of E_a,3 - E _a,1J.mol^–1 activation energy associated with k₁ - E _a,3J.mol^–1 activation energy associated with k₃ - E _d enzyme in deactivated form - ES enzyme/substrate complex - k ₁ s^–1 kinetic constant associated with the enzyme-catalyzed transformation of substrate - k _1,0 s^–1 preexponential factor associated with k₁ - k ₂ mol^–1.m³s^–1 kinetic constant associated with the binding of substrate to the enzyme - k _–2 s^–1 kinetic constant associated with the dissociation of the enzyme/substrate complex - K _2,0 mol.m^–3 constant value of K₂ - K _2,0 ^* dimensionless counterpart of K_2,0 - k ₃ s^–1 kinetic constant associated with the deactivation of enzyme - k _3,0 s^–1 preexponential factor associated with k₃ - k _3,0 ^* dimensionless counterpart of k_3,0 - P product - R J.K^–1.mol^–1 ideal gas constant - S substrate - t s time since start-up of reaction - T K absolute temperature - T ^* dimensionless absolute temperature - T _i,opt ^* optimal dimensionless isothermal temperature of operation - T _opt ^* optimal dimensionless temperature of operation - t _b s time of a batch - t _b ^* dimensionless counterpart of t_b - t _b,min ^* minimum value of the dimensionless counterpart of t_b Greek Symbols dimensionless counterpart of C_E,0 - dimensionless counterpart of C_E,b - dummmy variable of integration - dummy variable of integration - auxiliary dimensionless variable - ^* dimensionless variation of k₁ with temperature - _i ^* dimensionless value of k₁ under isothermal conditions - _opt ^* optimal dimensionless variation of k₁ with temperature     

Effect of muscle length on cross-bridge kinetics in intact cardiac trabeculae at body temperature

Dynamic force generation in cardiac muscle, which determines cardiac pumping activity, depends on both the number of sarcomeric cross-bridges and on their cycling kinetics. The Frank–Starling mechanism dictates that cardiac force development increases with increasing cardiac muscle length (corresponding to increased ventricular volume). It is, however, unclear to what extent this increase in cardiac muscle length affects the rate of cross-bridge cycling. Previous studies using permeabilized cardiac preparations, sub-physiological temperatures, or both have obtained conflicting results. Here, we developed a protocol that allowed us to reliably and reproducibly measure the rate of tension redevelopment (k_tr; which depends on the rate of cross-bridge cycling) in intact trabeculae at body temperature. Using K⁺ contractures to induce a tonic level of force, we showed the k_tr was slower in rabbit muscle (which contains predominantly β myosin) than in rat muscle (which contains predominantly α myosin). Analyses of k_tr in rat muscle at optimal length (L_opt) and 90% of optimal length (L₉₀) revealed that k_tr was significantly slower at L_opt (27.7 ± 3.3 and 27.8 ± 3.0 s⁻¹ in duplicate analyses) than at L₉₀ (45.1 ± 7.6 and 47.5 ± 9.2 s⁻¹). We therefore show that k_tr can be measured in intact rat and rabbit cardiac trabeculae, and that the k_tr decreases when muscles are stretched to their optimal length under near-physiological conditions, indicating that the Frank–Starling mechanism not only increases force but also affects cross-bridge cycling kinetics.     

Relationship of rapid light curves of variable fluorescence to photoacclimation and non-photochemical quenching in a benthic diatom

The response of rapid light–response curves (RLCs) of variable fluorescence to changes in short- and long-term photoacclimation status was studied in an estuarine benthic diatom. The diatom Nitzschia palea was grown under low- (LL, 20 μmol m⁻² s⁻¹) and high-light (HL, 400 μmol m⁻² s⁻¹) conditions, with the purpose of characterising the effects of long-term photoacclimation on (i) steady-state light–response curves (LC) of relative electron transport rate, rETR, (ii) the response of RLCs to changes in ambient irradiance (E, the irradiance to which the sample is acclimated to immediately before the RLCs), (iii) the relationship of RLCs to LC parameters and non-photochemical quenching (NPQ). Photoacclimation to LL and HL conditions induced distinct light–response patterns of rETR and NPQ. Higher growth light resulted in rETR vs. E curves with lower initial slopes (α, 0.591 μmol⁻¹ m² s vs. 0.661 μmol⁻¹ m² s, for HL and LL, respectively) and markedly higher maximum rates (rETR_m, 95.9 vs. 29.3), reached under higher E levels (higher light-saturation coefficient, E_k: 162.4 μmol m⁻² s⁻¹ vs. 44.3 μmol m⁻² s⁻¹). Acclimation to HL induced bi-phasic NPQ vs. E curves, with minimum values reached under low E levels (15–25 μmol m⁻² s⁻¹) and not on dark-acclimated samples. The response of RLCs to changes in ambient irradiance varied with the long-term photoacclimation status of the samples. The initial slope, α_RLC, decreased monotonically with E in LL cultures, from 0.68 to 0.25 μmol⁻¹ m² s, while varied bi-phasically in HL-acclimated samples. Typically, α_RLC of HL cultures increased under low E, reaching a maximum of 0.61 μmol⁻¹ m² s under 25–55 μmol m⁻² s⁻¹, and decreased gradually under higher E levels to 0.25 μmol⁻¹ m² s. RLC maximum rETR, rETR_m,RLC, and saturation coefficient E_k,RLC, increased with E following a saturation-like pattern, with the HL cultures presenting markedly higher values for all the E range (maximum rETR_m,RLC values were 108.6 and 33.4 for HL and LL cultures, respectively). An inverse relationship was consistently found between α_RLC and NPQ, both on LL and HL cultures, causing strong correlations (P < 0.001 in all cases) between NPQ and the high light-induced decrease of α_RLC, Δα_RLC. RLCs were confirmed to also provide information on the long-term photoacclimation status, as significant correlations (P < 0.001 both for HL and LL cultures) were verified between E_k and an index based on RLC parameters, Ê_k, both for LL and HL cultures. These results reinforce the usefulness of RLCs as a tool for inferring on the short- and long-term photoacclimation status of samples with different long-term light histories, through the estimation of LC parameters and the monitoring of NPQ levels.     

Diversity of Cytochrome bc Complexes: Example of the Rieske Protein in Green Sulfur Bacteria

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (E_m) of +160 mV in the pH range of 6 to 7.7 and a decreasing E_m (−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its E_m-versus-pH curve, in green sulfur bacteria are discussed.     

How performance of a recycle plug flow reactor is optimized for protease-driven autocatalytic reactions

The balance equations to a continuous plug flow recycle reactor performing a protease-mediated autocatalytic reaction were analytically developed under the assumption of a Michaelis-Menten type of mechanism. The necessary condition for the recycle ratio that leads to the minimum reactor volume was set up and the resulting expression was numerically solved for the optimum ratio of recycle, R _opt, as a function of the desired conversion of reactant (or pro-enzyme) into product (or enzyme), X _PE, for selected values of the relevant dimensionless parameters, i.e. the ratio of total concentrations of enzyme and product moieties, γ, and the ratio of the Michaelis-Menten parameter to the total concentration of product moiety, K _m ^*. It was numerically proven that variation of K _m ^* by five orders of magnitude centered around unity did not affect R _opt to a significant extent, whereas an empirical expression for R _opt versus γ and X _PE could be found and successfully fitted to the results of several numerical runs.     

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo

The equilibrium oxidation-reduction mipoint potential (E_m) of isolated Rhodopseudomonas sphaeroides cytochrome c₂ exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197–208). However, as with mammalian cytochrome c (Brandt, K.G., Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180–4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the E_m of cytochrome c₂ before and after the conformational change. The E_m of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium E_m is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c₂ to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent E_m from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c₂ in vivo involve protons.     

Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine

Correlation of hepatitis C virus (HCV) spontaneous resolution with Th1 and CD8⁺CTL responses during natural infection implies the potentiality of poly-CTL-epitopic HCV vaccines. We recently reported in silico design and construction of DNA vaccines (pcPOL-plasmids) harboring HCV CTL epitopes. Herein, we provide data of mice immunization by pcPOL, (encoding; core_132-142 [C], E2_405-414 [E₄], E2_614-622 [E₆] and NS3_1406-1415 [N] CD8⁺CTL epitopes as CE₄E₆N polytope) and its HBsAg-fused counterpart (pcHPOL), compared to the adjuvant-formulated (Montanide + CpG) CE₄E₆N synthetic-peptide immunization. All vaccinated groups developed different levels of cellular responses, however, only the pcHPOL-immunized mice elicited strong CTLs and IFN-γ-secreting cells that were further augmented towards a Th1 response and partial tumor protection by DNA-prime/peptide-boosting regimen. Priming with HBsAg alone could not afford its augmenting effect indicating the importance of priming by polytope itself. Hence, fusion of immunocarriers like HBsAg conjoined with DNA-prime/peptide-boost immunization regimen seems a strategy to enhance the epitope-specific immune responses towards poly-CTL-epitopic vaccines.     

The interplay of O-H?O hydrogen bonding in the generation of three new supramolecular complexes of Cu, Ni and Co: Syntheses, characterization and structural aspects

Three new coordination complexes, [Cu(L¹)(H₂O)] (1), [Ni(L²)₂]·CH₃CN (2) and [Co(HL³)(L³)] (3) [where H₂L¹, N,N′-bis(3-methoxysalicylidenimino)-1,3-diamino-propane; HL², 2-((E)-(1,3-dihydroxy-2-methylpropan-2-ylimino)methyl)phenol; H₂L³, 2-((E)-(2-hydroxyethylimino)methyl)-4-bromophenol] have been synthesized and systematically characterized by elemental analyses, FT-IR, electronic spectroscopy, cyclic voltammetry and thermogravimetric analyses. Single crystal X-ray diffraction studies confirm that the metal center in complex 1 has distorted square-pyramidal geometry while it is distorted octahedral in the other two complexes. In all the complexes O-H?O hydrogen bondings assemble the molecular units leading to ordered supramolecular architectures. While both complexes 1 and 2 form infinite one-dimensional arrays through the self organisation of hydrogen bonded ring motifs, complex 3 is a unique star-shaped cyclic hexamer generated through intermolecular hydrogen bonding.     

Differences between CTP and ATP as substrates for the (Na + K)-ATPase

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K_m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E₂ conformational state of the enzyme, K⁺, Mg²⁺, and Mn₂⁺, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E₁ conformational state of the enzyme, K⁺, Mg²⁺, and Mn²⁺, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E₁ and E₂ conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E₂ state. This reaction is better stimulated by CTP plus Na⁺ than by ATP plus Na⁺; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E₂ state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E₂ to E₁, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E₂ over the E₁ conformational state.     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K₂)Mg–MgF₄, an E2∙Pi-like state capable to occlude K⁺ (or Rb⁺). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb⁺ with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1–E2 conformational transition and the kinetics of Rb⁺ exchange between the medium and the E2(Rb₂)Mg–MgF₄ state. Our results show that both in the absence and in the presence of Rb⁺, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg–MgF₄ complex, that is unable to shift to E1 upon addition of Na⁺. The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg–MgF₄ formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb⁺ caused by Mg^2 +. Na⁺-induced release of Rb⁺ from E2(Rb₂)Mg–MgF₄ occurs at the same rate as from E2(Rb₂) but is insensitive to ADP. The rate of Rb⁺ occlusion into the E2Mg–MgF₄ state is 5–8 times lower than that described for the E2Mg–vanadate complex. Since the E2Mg–MgF₄ and E2Mg–vanadate complexes represent different intermediates in the E2-P → E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.     

Homogeneous green catalysts for olefin oxidation by mono oxovanadium(V) complexes of hydrazone Schiff base ligands

Three mono oxovanadium(V) complexes of tridentate Schiff base ligands [VO(OMe)L¹] (1), [VO(OMe)L²] (2) and [VO(OMe)L³] (3) obtained by monocondensation of 3-hydroxy-2-naphthohydrazide and aromatic o-hydroxyaldehydes have been synthesized (H₂L¹ = (E)-3-hydroxy-N′-(2-hydroxy-3-methoxybenzylidene)-2-naphthohydrazide, H₂L² = (E)-3-hydroxy-N′-(2-hydroxybenzylidene)-2-naphthohydrazide and H₂L³ = (E)-N′-(5-bromo-2-hydroxybenzylidene)-3-hydroxy-2-naphthohydrazide). The complexes were characterized by spectroscopic methods in the solid state (IR) and in solution (UV-Vis, ¹H NMR). Single crystal X-ray analyses were performed with 1 and 2. The catalytic potential of these complexes has been tested for the oxidation of cyclooctene using H₂O₂ as the terminal oxidant. The effects of various parameters including the molar ratio of oxidant to substrate, the temperature, and the solvent have been studied. The catalyst 2 showed the most powerful catalytic activity in oxidation of various terminal, cyclic and phenyl substituted olefins. Excellent conversions have been obtained for the oxidation of cyclic and bicyclic olefins.     

Studies on synthesis, characterization, and G-quadruplex binding of Ru(II) complexes containing two dppz ligands

In this work, the interaction between the guanine-rich single-strand oligomer AG₃(T₂AG₃)₃ quadruplex and two Ru(II) complexes, [Ru(L¹)(dppz)₂](PF₆)₄ (1) and [Ru(L²)(dppz)₂](PF₆)₄ (2) (L¹ = 5,5′-di(1-(trimethylammonio)methyl)-2,2′-dipyridyl cation, L² = 5,5′-di(1-(triethylammonio)methyl)-2,2′-dipyridyl cation, dppz = dipyrido[3,2-a:2′,3′-c] phenazine), has been studied by UV-Visible, fluorescence, DNA melting, and circular dichroism in K⁺ buffer. The two complexes after binding to G-quadruplex have shown different DNA stability and fluorescence enhancement. The results show that both complexes can induce the stabilization of quadruplex DNA. ΔT_m values of complexes 1 and 2 at [Ru]/[DNA] ratio of 1:1 were 9.4 and 7.0, respectively. Binding stoichiometry along with the quadruplex was investigated through a luminescence-based Job plot. The major inflection points for complexes 1 and 2 were 0.49 and 0.46, respectively. The data were consistent with the binding mode at a [quadruplex]/[complex] ratio of 1:1. In addition, the conformation of G-quadruplex was not changed by the complexes at the high ionic strength of K⁺ buffer.     

Partitioning of latent heat flux at a northern peatland

The partitioning of latent heat flux (Q_E) to vascular plant and moss surface components was assessed for a Sphagnum-dominated bog with a hummock–hollow surface having a sparse canopy of low shrubs. Results from porometry and eddy covariance measurements of Q_E showed evaporation from the moss surface ranged from greater than 50% of total Q_E early in the growing season to less than 20% after a dry period toward the end of the growing season. Both soil moisture and vapour pressure deficit (D_a) affected this partitioning with drier moss and peat, lower water table, and smaller D_a all reducing moss Q_E. Daily maximum moss Q_E ranged from greater than 200 W m⁻² early in the growing season to less than 100 W m⁻² during a dry period. In contrast, vascular contribution to total Q_E increased over the season from a daily maximum of about 150 W m⁻² to 250 W m⁻² due to increase in leaf area by leaf replacement and emergence and to drying of the moss surface. Porometry results showed average daily maximum conductance from bog shrubs was near 8 mm s⁻¹. These conductance values were smaller than those reported for vascular plants from more nutrient-rich wetlands. The effect of increases in D_a on vascular Q_E were moderated by decreases in stomatal conductance. At constant available energy, vascular leaf conductance was reduced by as much as 2 mm s⁻¹ and moss surface conductance was enhanced by up to 3 mm s⁻¹ by large D_a. Considering vascular and non-vascular water transport characteristics and frequency of water table position and given the observed variations of Q_E partitioning with water table location and moss and peat water content, it is suggested that modelling efforts focus on how dry hummocks and wet hollows each contribute to Q_E, especially as related to D_a and soil moisture dynamics.